Online reference calibration

ABSTRACT

An online calibration system for an electrochemical sensor. The calibration system comprises a calibration electrode coupled with a redox species, where the redox species is configured to control a pH of a reference solution local to the calibration electrode, such that when a voltammetric signal is applied to the calibration electrode the output generated from the calibration system is determined by the local environment pH. The output signal from the calibration system is used to calibrate a reference potential generated by a reference system of the electrochemical sensor to correct for drift in the reference potential when the electrochemical sensor is being used. The calibration electrode may be disposed in a reference cell of the electrochemical sensor.

This application is a U.S. National Stage of International ApplicationNo. PCT/EP2018/077797, filed Oct. 11, 2018, which claims the benefit ofGreat Britain Patent Application No. 1716652.1 filed on Oct. 11, 2017,Great Britain Patent Application No. 1716660.4 filed on Oct. 11, 2017,and International Application No. PCT/IB2018/000097, filed Feb. 12,2018; the entire disclosures of all are hereby incorporated by referencein their entireties into the present disclosure for all purposes.

BACKGROUND

The present disclosure describes an online calibration system for anelectrochemical sensor. The online calibration system comprises acalibration electrode configured to be deployed in a reference cell ofthe electrochemical sensor and to contact a reference solution therein.The calibration electrode comprises a redox species that is sensitive topH/hydrogen ion concentration and is configured to set a pH of thereference solution local to the calibration electrode, such that when anelectrochemical/voltammetric signal is applied to the calibrationelectrode, the calibration electrode generates anelectrochemical/voltammetric response that has a constant feature, suchas a peak potential, corresponding to the set pH of the referencesolution local to the calibration electrode. The reference potential ofthe electrochemical sensor produced by the reference cell is calibratedby comparing a difference between the constant feature of theelectrochemical/voltammetric response of the calibration electrode andthe reference potential while the electrochemical sensor is deployedand/or being used. By repeatedly processing this difference, thereference potential can be calibrated online, without manualintervention.

In general there are three types of electrochemical sensors,voltammetric sensors, potentiometric sensors and/or amperometricsensors.

Amperometric sensors normally comprise at least a first electrode and areference electrode. In use, a voltage is applied between the firstelectrode and the reference electrode and the resulting current betweenthe first electrode and the reference electrode is measured. The currentis produced by oxidation/reduction of a chemical species that the sensoris configured to detect and the measured current is indicative of theconcentration of the chemical species.

For the measured current to be meaningful, the potential applied betweenthe first electrode and the reference electrode needs to be a knownpotential. In many amperometric sensors, the reference electrode iscontacted with a reference solution, which is a solution containingchloride ions, such as potassium chloride (KCl) solution, a sodiumchloride (NaCl) solution and/or the like, to maintain the potential ofthe reference electrode as a constant. However, in use, the referencesolution may become diluted, reducing the concentration of Cl ions,contaminated and, as a result, the potential applied between the firstelectrode and the reference electrode may drift, reducing the accuracyof the reference system.

Voltammetric/potentiometric sensors are some of the most common types ofelectrochemical sensor. Potentiometric sensors are the basis for glasselectrodes (used for measuring pH, sodium (Na+), potassium (K+), lithium(Li+) and the like), solid membrane electrodes (based on the chemicalprocess AgX for X−), liquid membrane electrodes (e.g. containing aligand for M+ complexation and used in calcium (Ca+) and K+ sensors),pH-meter-based gas detectors (e.g. carbon dioxide (CO2) sensors, ammonia(NH3) sensors etc.) and some solid oxide sensors (e.g. zirconia-basedoxygen (O2) sensors). Voltammetric sensors may be used to measure pH,glucose, oxygen, hydrogen sulphide, for biosensing, for pharmaceuticalsensing and/or the like. Potentiometric/voltammetric sensors measure apotential difference between an electrode or environment that issensitive to the desired analyte and an electrode or environment that isinsensitive to the analyte. In such sensors, an electrode or environmentthat is sensitive to the analyte is known as the sensing electrode andthe electrode or environment that is insensitive to the analyte is knownas the reference electrode.

Ion-sensitive field-effect transistors (ISFETs) are a new generation ofsolid state potentiometric sensor. In an ISFET, the sensing electrode isreplaced with ion selective field effect transmitter, which measures avoltage between a source and a drain that is dependent on aconcentration of an analyte in a solution being measured. To processproperties of an analyte this source-drain voltage is measured againstan output from a reference electrode housed in a well-definedenvironment.

For both traditional potentiometric and ISFET sensors, significant workhas been performed to developing novel sensing electrode to measuredifferent analytes/ions and/or to improve the accuracy/sensitivity forsensing of an ion/analyte. This work has resulted in the development ofa range of commercial sensors that can achieve the desired selectivityand sensitivity to measure a range of analytes/ions.

However, despite the abundance and importance of potentiometric andISFET sensors, operation of the sensors is problematic due toinstabilities in the reference electrode. Typically, the referenceelectrode comprises a silver/silver chloride (Ag/AgCl) electrode that isheld within a defined environment, containing a reference solution,behind a porous frit that allows for electrical conductivity to thesolution that the sensor is measuring.

For a typical silver-silver chloride (Ag/AgCl) reference system, theAg/AgCl electrode is stored in a solution with known properties when notin operation as the electrode can dry out when held in a dry atmosphere.Also, the Ag/AgCl sensor requires periodic recalibration due to driftsin the reference electrode potential because the chemistry of theelectrode may change and/or ions may pass through the frit and perturbthe environment. Furthermore, potentiometric systems typically operatein a continuous single point measurement mode, which is a mode where aknown current is held between the reference and sensing electrode(typically 0) and the potential difference is constantly measured.

The very nature of this measurement means that any drift in thereference electrode during use is difficult to monitor, as there is noway to detect the drift and as a result the drift may be wronglyassigned to variations in the analyte concentration; typicallycorrections for drift are made after use when the sensor is recalibratedand any changes in the calibration shown before and after use areextrapolated linearly and the data from the sensor is revised inaccordance with the extrapolations. However, such assumptions mean thatthe sensor may provide inaccurate results and sensor operation requiresmanual input. For example, for regulatory purposes it is necessary to beable to calibrate sensors requiring use of a standard extrapolationprocess to ensure for standardized measurements. More importantly, thecalibration requirement means that the sensors require constantcalibration, which may be expensive and or require user intervention,meaning the sensor cannot be accurately used in an online process and/orautonomously.

Several researchers have taken on the challenge of increasing thestability of the reference electrode and numerous methods have beenproposed to overcome the issue.

U.S. Pat. No. 5,419,826 describes an ion-selective reference probeadapted for use with potentiometric measurement systems. The referenceprobe is non-chloride based and employs a specially adapted electrolyte,which is reversible with regard to ionic activity.

United States Patent Publication Number 20030024812 discloses a solidstate electrochemical reference system, containing two or moreelectrodes, wherein the half-cell potential of at least one electrode isdetermined by the concentration of a specific ion anticipated to bepresent in all test solutions. The ion concentration measured in thecell by a first electroanalytical technique does not depend on a knownreference electrode potential, such that said electrode, its half-cellpotential being calculable from the measured ion concentration, canserve as a reference electrode in one or more subsequentelectroanalytical techniques that do depend on a known referenceelectrode potential, said subsequent technique or techniques beingcarried out in the same cell.

U.S. Pat. No. 6,398,931 details an improved combination ion-selectiveelectrode apparatus comprising an electrode body, a reference electrode,and an ion-sensing electrode. The reference electrode comprises anion-permeable junction and a removable membrane cap contains anion-selective membrane. The membrane cap can be removed from theion-selective electrode apparatus without endangering the integrity ofthe reference electrode and is distinct from the ion-permeable junction.

European Patent Number 2 932 249 describes a reference electrode for anelectrochemical sensor that comprises an inner reference element, whereinner reference element has been embedded into a solid electrochemicallyactive composite material.

U.S. Pat. No. 7,462,267 describes a reference electrode consisting of ametal in contact with an electrolyte containing an anion or cation whoseconcentration in part determines the redox potential of the electrode.This electrolyte contains a polyelectrolyte that partially andreversibly binds the chemical cation or anion thus lowering the freeconcentration of the cation or anion compared to the osmotic pressure ofthe same concentration of cation or anion if present as a simple salt.The polyelectrolyte can be anionic or cationic depending on thechemistry of the redox electrode and a thickener may also be added tothe electrolyte.

However, to-date, the techniques for stabilizing/calibrating thereference electrode are complex, require manual intervention and regularmaintenance, and use potentiometric operation, which may compound/maskstability errors. Because of the need to recalibrate the referencesystem of most electrochemical sensors, the sensors cannot be accuratelyused for long duration operation, cannot be used in anautonomous/networked system, and require costly manual calibration.

SUMMARY

In embodiments of the present disclosure, a calibration system isprovided that comprises a calibration electrode, which may comprise aworking electrode, that is used to make a voltammetric/electrochemicalmeasurement and this measurement is used to verify/calibrate theelectrochemical potential of a reference electrode of a potentiometricsensor. In this way, the calibration system can provide a correction toany drift in the reference electrode, without manual intervention.Moreover, with respect to ion selective sensors and/or amperometricsensors, because the calibration measurement is anelectrochemical/voltammetric measurement the drift is not masked by,and/or is independent of potentiometric operation of the electrochemicalsensor.

For purposes of this disclosure, the term electrochemical andvoltammetric are used interchangeably with respect to applying apotential to a redox species. For example, in embodiments of the presentdisclosure, a potential is swept across the calibration electrode andthis swept potential may be referred to as a voltammetric orelectrochemical signal or as a voltammetric or electrochemical sweep.The term ‘voltammetric’ is commonly used to a potential sweep and theterm ‘electrochemical’ is used to refer to a potential applied to achemical species and/or the resulting electrical signal generated by theapplication of the potential since it is an electrochemical process.

For purposes of this disclosure, the calibration electrode is configuredto be a working electrode in an electrochemical cell.

For purposes of this disclosure, the other electrodes in theelectrochemical cell with the calibration electrode may be referred toas counter electrodes, reference electrodes and/or auxiliary electrodes.

In embodiments of the present disclosure, the calibration electrode isused in an electrochemical/voltammetric system, which may comprise acounter and a reference/auxiliary electrode, to generate a voltammetricresponse. The electrochemical/voltammetric response of the calibrationelectrode is used to calibrate a reference system of the electrochemicalsensor that generates a reference potential, where the referencepotential is produced by a reference system comprising a reference cell.

In embodiments of the present disclosure, the calibration electrodecomprises a redox active species (a species that undergoesoxidation/reduction when a current/potential is applied) that issensitive to pH (the oxidation/reduction current changing with respectto pH/hydrogen ion concentration) and configured to control the pH of areference solution local to the calibration electrode. The redox activespecies may control the pH of the local environment proximal to thecalibration electrode in the following ways. First, the redox activespecies may comprise either an acid or a base and/or contain acid orbase moieties. In this way, the acidic or basic nature of the redoxactive species sets the pH of the local environment of the referencebecause the reference solution, having a low buffering capacity, cannotbuffer the effect of the acidic or basic redox active species. In thesecond case, the redox active species may trigger the pH of the localenvironment of the reference solution by consuming or donating protonsto the reference solution when a potential is applied to the redoxactive species. Again, because the reference solution cannot buffer theeffect of the proton consumption or donation, the redox active speciessets the local pH of the reference solution proximal to the calibrationelectrode.

The reference solution is the solution contained in a reference chamberof the electrochemical sensor, and as described above, normallycomprises potassium chloride solution or the like. The referencesolution provides a saturated chloride ion solution to maintain aconstant reference potential. Potassium chloride, sodium chloride andthe like are bufferless, low buffering capacity solutions. Because thesereference solutions have a low buffering capacity or contain no buffer,the redox species will affect the local environment of the referencesolution proximal to the redox species as there is no buffer/bufferingcapacity to buffer the local effect of the redox species. In embodimentsof the present disclosure, redox active species sensitive to pH maycomprise: a chemistry that affects hydrogen ion concentration (consumesor produces protons during oxidation/reduction), a base, an acid, basemoieties, acid moieties or the like to set a local pH of the referencesolution to a value greater or less than a pH of 7. By way of example,the redox species may comprise anthraquinone, ferrocene, salicylic acidor the like. In fact, all redox active systems that are sensitive to pHwill set the local pH of a low buffering capacity solution duringoxidation/reduction, and this effect can only be prevented by using aredox active system that essentially negates the effect. Whileembodiments of the present disclosure can use a redox species that setsthe local pH of the reference solution to 7, this is not a preferredembodiment as it may complicate calibration processing.

In embodiments of the present disclosure, anelectrochemical/voltammetric sweep is applied to the calibration(working) electrode to generate an electrochemical/voltammetric responsefrom the calibration electrode, where the electrochemical/voltammetricresponse comprises oxidation/reduction of the redox active species. Thevoltammetric response includes singularities/peaks in theoxidation/reduction current and a potential corresponding to thesepeaks/singularities is known as the peak potential. The peak potentialfor redox active species sensitive to pH is set by the pH of thesolution that the calibration electrode is contacting.

In embodiments of the present disclosure, the pH of the referencesolution local to the calibration electrode is set by the redox activespecies. As such, the peak potential generated by the calibrationelectrode is a constant value. In operation, when anelectrochemical/voltammetric sweep is applied to the redox activespecies, the redox active species undergoes oxidation/reduction. Duringthis oxidation/reduction, the redox active species donates or consumesprotons, depending upon the chemistry of the redox active species, andbecause the contacted solution has a low buffer capacity this effect isnot buffered by the solution so the redox species produces anelectrochemical/voltammetric response that is governed by theproton/hydrogen ion concentration proximal to the calibration electrodethat it has generated.

In embodiments of the present disclosure this constant peak potential,and/or a related potential in the electrochemical/voltammetric response,is used as a reference/calibration value to calibrate the referencepotential of the electrochemical sensor. As such, in embodiments of thepresent disclosure, the calibration/working electrode may be sweptperiodically, while the electrochemical is deployed/in use and thedetermined peak potential used to calibrate the reference potential,without manual intervention.

In some embodiments, calibration of the reference electrode potential isprovided by measuring a difference between the potential of thereference electrode and a peak potential or the like produced by thecalibration electrode, where any changes in the difference are used tocorrect/calibrate the output from the sensor since the peak potential isa constant potential and any changes will be due to drift in thereference potential. Because the reference solution of mostelectrochemical sensors comprise chloride ions, the redox active speciesof the calibration electrode is selected such that it is insensitive tochloride ion concentration. In this way, the calibration electrode andthe peak potential generated by the calibration electrode is independentof the concentration of chloride ions of the reference solution.

Surprisingly, applicant has found from extensive testing that the effectof the redox active species controlling the local pH of the referencesolution, and the resulting constant peak potential generated byoxidation/reduction of the redox active species, is unperturbed by:large dilution of the reference solution (up to 50% dilution); presenceof acids or bases in the reference solution; presence of activechemistries in the reference solution; presence of carbonates, e.g. hardwater (carbonates are problematic as they affect the buffering capacityof the solution and thereby reduce the ability of the redox activespecies to set the local pH); and/or the like.

While peak potential, a peak in the electrochemical/voltammetric signalgenerated by the calibration electrode may be used to calibrate thereference potential, other potentials in theelectrochemical/voltammetric response, such as a change in direction ofthe generated sweep signal, a maximum rate of change on the sweep signalor other characteristic that can be ascertained by signal processing—maybe used to obtain a calibration potential of the calibration electrodeand this may then be used to calibrate the reference electrode. In someembodiments, multiple points of the electrochemical/voltammetric sweepresponse of the calibration electrode may be analysed to generate acalibration potential. Moreover, since the pH set by the redox activespecies in a reference solution is known, can be calculated, or can bedetermined from tests/experiments, the signal processor can analyze theelectrochemical/voltammetric response to an applied potential sweepusing this knowledge.

Since the voltammetric/electrochemical measurement is not apotentiometric measurement, unlike the measurement from the referenceelectrode, the measurement provides a truly independent calibration.Moreover, the voltammetric/electrochemical measurement may be madeperiodically reducing issues/maintenance requirements associated withcontinuous/high frequency measurements. Also, the calibration system maybe used with a robust reference system, such as an Ag/AgCl referencesystem and does not rely on potentiometrically measuring the presence ofan ion.

In embodiments of the present disclosure, the calibration electrodecomprises a redox active species that controls the local environment ofa reference solution of the electrochemical sensor proximal to theelectrode. This control of the local environment may in some embodimentsbe provided by contacting the electrode with a low buffer/low ionicstrength solution, such as water, seawater, sodium chloride solution,silver, potassium chloride solution and/or the like. In such, anenvironment, the calibration electrode, because of the low buffer/ionicstrength of the analyte, ‘sees’ an environment controlled by the redoxactive species itself. For example, a common redox active species forelectrochemical sensors, anthraquinone will measure a pH of about 10 or11 when a voltammetric signal is applied because the anthraquinone willconsume protons during reduction triggering a change in pH in the localenvironment that is measured by the sensor. This effect of the redoxspecies will occur for most redox active species, unless they arespecially configured, when the analyte contacted with the redox speciesis a low buffer/low ionic strength analyte

In other embodiments, a redox species containing acid groups, such assalicylic acid etc., or alkali groups, such as species containing aminegroups, acid groups will create an acidic or alkaline local environmentirrespective of the acidity/alkalinity of the fluid being sensed. Insuch embodiments, the local environment is controlled by theacidic/alkaline redox species even if the buffer/ionic strength of theanalyte contacting the redox species is not low. In some embodiments ofthe present invention, redox species with acid or alkali groups are usedto move the pH of the local environment away from a neutral reading, apH of 7, to provide a known reference potential output from thecalibration electrode when a voltammetric signal is applied to thecalibration electrode.

Such an approach can be utilized in all electrochemical systems whichrequire a stable reference electrode system. Potassium ion sensorsutilize a valinomycin modified membrane to provide the ion-selectiveresponse, in conjunction with a standard Ag/AgCl electrode. Thelongevity of such systems is often compromised by instability in thereference electrode. In such systems the addition of a calibration sweepsystem using an electrode with a controlled environment would obviatethe lifetime issues associated with the drift in the referenceelectrode.

Up until now, the effect of the redox species controlling the localenvironment has been identified as a weakness in electrochemical sensoroperation as it produces incorrect output from the sensor since thesensor measures properties of the local environment, which is controlledby the redox species, not the properties of the solution being tested.However as described herein, the effect provides an electrode that has aknown output, due to its control of the local environment, that may beused for calibration.

In sensors designed for use in low buffer/low ionic strength solutions,such as water/seawater or the like, the calibration electrode may becontacted directly with the low buffer/low ionic strength fluid and theredox species control the local environment to produce a known/stablepotential output from the calibration electrode. In sensors that may beused with fluids with unknown properties and/or high ionicstrength/buffer strength, the calibration electrode may be contactedwith a known analyte, such as an analyte kept behind a frit or the like,for example an aqueous solution with low ionic/buffer strength. In someembodiments, the calibration electrode may be contacted with the samefluid environment as the reference electrode, i.e., a reference solutionheld in a reservoir behind a frit that allows for electrical/ionconductivity with the solution being tested/analyzed.

In some embodiments, the calibration system may comprise an additionalelectrochemical cell that is placed inside an existing referenceelectrode chamber. In such an arrangement, the reference electrode inthe existing reference electrode chamber may be used as a referenceelectrode for the calibration system.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, similar components and/or features may have the samereference label. Further, various components of the same type may bedistinguished by following the reference label by a dash and a secondlabel that distinguishes among the similar components. If only the firstreference label is used in the specification, the description isapplicable to any one of the similar components having the same firstreference label irrespective of the second reference label.

FIG. 1A illustrates a glass electrode pH sensor with a referenceelectrode.

FIG. 1B is a schematic-type illustration of an electrochemical sensorcomprising a reference electrode/reference system.

FIG. 2A illustrates an electrochemical sensor comprising a calibrationsystem, in accordance with some embodiments of the present disclosure.

FIG. 2B illustrates an electrochemical sensor comprising a calibrationsystem, in accordance with some embodiments of the present disclosure.

FIG. 2C depicts an electrochemical sensor comprising a calibrationsystem, in accordance with some embodiments of the present disclosure,and outputs from the calibration system.

FIG. 3 is a flow-type illustration of a method for online calibration ofa reference system of an electrochemical sensor, in accordance with someembodiments of the present disclosure.

FIG. 4 is a graph that displays the response of AQ modified electrodesin pH 4, 7 and 10 buffers

FIG. 5A shows a four component system incorporating the solid electrode,the electroactive system, the polymeric layer and the bulk solution.

FIG. 5B shows a three component system comprising of the solid electrodein which the electroactive species is immobilized, a polymeric layer andthe bulk solution.

FIG. 6 is a graph that displays the Electrochemical response of Nafioncoated system in pH 4, 7 and 10 buffers.

FIG. 7 details the response of a) polyaniline, b) cellulose acetate andc) an electropolymerised salicylic acid coated electrode when placed in3M KCl and pH 10 solution.

FIG. 8 details the plot of peak potential against time showing the peakpotential measured for a first, 10th, 15th and 20th scan.

DESCRIPTION

The ensuing description provides some embodiment(s) of the invention,and is not intended to limit the scope, applicability or configurationof the invention or inventions. Various changes may be made in thefunction and arrangement of elements without departing from the scope ofthe invention as set forth herein. Some embodiments may be practicedwithout all the specific details. For example, circuits may be shown inblock diagrams in order not to obscure the embodiments in unnecessarydetail. In other instances, well-known circuits, processes, algorithms,structures and techniques may be shown without unnecessary detail inorder to avoid obscuring the embodiments.

Some embodiments may be described as a process which is depicted as aflowchart, a flow diagram, a data flow diagram, a structure diagram, ora block diagram. Although a flowchart may describe the operations as asequential process, many of the operations can be performed in parallelor concurrently. In addition, the order of the operations may bere-arranged. A process is terminated when its operations are completed,but could have additional steps not included in the figure and may startor end at any step or block. A process may correspond to a method, afunction, a procedure, a subroutine, a subprogram, etc. When a processcorresponds to a function, its termination corresponds to a return ofthe function to the calling function or the main function.

Moreover, as disclosed herein, the term “storage medium” may representone or more devices for storing data, including read only memory (ROM),random access memory (RAM), magnetic RAM, core memory, magnetic diskstorage mediums, optical storage mediums, flash memory devices and/orother machine readable mediums for storing information. The term“computer-readable medium” includes, but is not limited to portable orfixed storage devices, optical storage devices, wireless channels andvarious other mediums capable of storing, containing or carryinginstruction(s) and/or data.

Furthermore, embodiments may be implemented by hardware, software,firmware, middleware, microcode, hardware description languages or anycombination thereof. When implemented in software, firmware, middlewareor microcode, the program code or code segments to perform the necessarytasks may be stored in a machine readable medium such as storage medium.A processor(s) may perform the necessary tasks. A code segment mayrepresent a procedure, a function, a subprogram, a program, a routine, asubroutine, a module, a software package, a class or any combination ofinstructions, data structures or program statements. A code segment maybe coupled to another code segment or a hardware circuit by passingand/or receiving information, data, arguments, parameters or memorycontents. Information, arguments, parameters, data, etc. may be passed,forwarded or transmitted via any suitable means including memorysharing, message passing, token passing, network transmission, etc.

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings and figures. In thefollowing detailed description, numerous specific details are set forthin order to provide a thorough understanding of the subject matterherein. However, it will be apparent to one of ordinary skill in the artthat the subject matter may be practiced without these specific details.In other instances, well known methods, procedures, components, andsystems have not been described in detail so as not to unnecessarilyobscure features of the embodiments. In the following description, itshould be understood that features of one embodiment may be used incombination with features from another embodiment where the features ofthe different embodiment are not incompatible.

It will also be understood that, although the terms first, second, etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. For example, a first object or step could betermed a second object or step, and, similarly, a second object or stepcould be termed a first object or step. The first object or step, andthe second object or step, are both objects or steps, respectively, butthey are not to be considered the same object or step.

The terminology used in the description of the disclosure herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the subject matter. As used in thisdescription and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. It will also be understood that theterm “and/or” as used herein refers to and encompasses any and allpossible combinations of one or more of the associated listed items. Itwill be further understood that the terms “includes,” “including,”“comprises,” and/or “comprising,” when used in this specification,specify the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof.

As used herein, the term “if” may be construed to mean “when” or “upon”or “in response to determining” or “in response to detecting”, dependingon the context. Similarly, the phrase “if it is determined” or “if [astated condition or event] is detected” may be construed to mean “upondetermining” or “in response to determining” or “upon detecting [thestated condition or event]” or “in response to detecting [the statedcondition or event],” depending on the context.

For the purposes of this disclosure the following terms have thefollowing meaning.

A “redox-species” is a compound or composition that may be oxidized andreduced. “Redox activity” refers to either or both of those processes.

A “redox sensitive species” is redox-species that is sensitive orsubstantially sensitive to the presence or concentration of an analytein a sample within those user-defined application-specific tolerances.“Substantially sensitive” to an analyte is used to mean sensitive withinthe tolerances required for a given application, as those tolerances aredefined by an end user.

A “redox-active material” is a compound or composition that may beoxidized and reduced. “Redox activity” refers to either or both of thoseprocesses.

A “reference electrode” (RE) is an electrode used to establish thepotential difference applied to the working electrode (WE). ConventionalREs have a certain fixed chemical composition and therefore a fixedelectrochemical potential, thus allowing measurement of the potentialdifference applied to the WE in a known, controlled manner. An REtypically comprises two halves of a redox couple in contact with anelectrolyte of fixed chemical composition and ionic strength. Becauseboth halves of the redox couple are present and the composition of allthe species involved is fixed, the system is maintained at equilibrium,and the potential drop (i.e., the measured voltage) across theelectrode-electrolyte interface of the RE is then thermodynamicallyfixed and constant. For example a commonly used RE system is theAg/AgCl/KCl system with a defined and constant concentration of KCl. Thetwo half-cell reactions are therefore: Ag++e−→Ag; and AgCl+e−→Ag+Cl—.The overall cell reaction is therefore: AgCl→Ag++Cl— for which theNernst equilibrium potential is given as: E=E0−(RT/F)*ln [Cl—], where Eis the measured RE potential, E0 is the standard potential of theAg/AgCl couple vs. the standard hydrogen electrode with all species atunit activity (by convention the standard hydrogen electrode is definedas having a potential of 0.0V); and R, T, and F are the universal gasconstant, temperature, and Faraday constant, respectively, inappropriate units. Hence, the potential of this system depends only onthe concentration (more strictly speaking the activity) of Cl— ionpresent, which, if this is fixed, provides a stable, fixed potential.Many other RE systems are known in the art. It is imperative that thecomposition of the RE remains constant, and hence almost no currentshould be passed through the RE (otherwise electrolysis will occur andthe composition of the RE will change), which necessitates the use of athird electrode, the counter electrode (CE), to complete the circuit.However, two-electrode configurations can be used in the special casewhere the WE is a microelectrode, having at least one dimensiontypically smaller than 100 micrometers. In this case, the currentspassed at the WE are small, and therefore a two-electrode cell can beused with a RE, but without the need for a CE.

A “sensor” is an electrode or collection of electrodes that generates asignal in response to the presence of an analyte. A sensor can include,for example, a working electrode, a counter-electrode and a referenceelectrode (either a conventional reference electrode or a pseudoreference electrode). A sensor can include, for example, a workingelectrode, a counter electrode and an analyte-insensitive electrode.

An “electrode” is a component of a sensor and may comprise a metal,carbon and/or the like. A variety of carbon substrates are suitable foruse as substrate material in the electrodes of the present invention,including but not limited to carbon allotropes such as graphites,including pyrolytic graphite and isotropic graphite, amorphous carbon,carbon black, single- or multi-walled carbon nanotubes, graphene, glassycarbon, boron-doped diamond, pyrolyzed photoresist films, and othersknown in the art.

A “working electrode” is the electrode at which the electrochemicalprocess for detecting the analyte of interest occurs. In a sensor, theworking electrode may be sensitive to one or more analyte(s) in the testsample, or it may be chemically modified with analyte sensitivespecies/materials. The electrochemical response of the working electrodeis measured after some perturbation to the system under study has beenapplied. For example, the perturbation may be the application of apotential difference to the working electrode that induces electrontransfer to occur, and the resulting current at the working electrode isthen recorded as a function of the applied potential (voltammetricmode). This example of mode of operation is illustrative and notexhaustive, as many other modes are known in the art. The workingelectrode may comprise a redox species that can undergo a reversibleelectrochemical redox reaction dependent upon the concentration of ananalyte (hydrogen ions for a pH meter; other analytes for other analytesensing devices) in a sample solution and an applied electricalpotential. For example, where there is a high concentration of hydrogenions present in a sample solution, the redox reaction occurs at a lowerpotential. Conversely, where there is a low concentration of hydrogenions present in a sample solution, the redox reaction occurs at a higherpotential. The relationship between these characteristic potentials andthe sample solution pH is a function of the chemical identity of theredox species. An algorithm converts electrical potential to pH value toprovide a means of determining the pH of an unknown sample.

FIG. 1A illustrates a glass electrode pH sensor. Glass electrodes aresome of the most common electrochemical sensors. The glass electrode isan example of an ion selective electrode/sensor.

Glass electrode pH sensors are some of the most ubiquitouselectrochemical sensors, since they use a well-established technology tomeasure a fundamental property, pH. The glass electrode comprises aninternal electrode 10 contained in a measurement chamber 12. The glasselectrode is disposed in a test solution 20 and the internal electrode10 is in electrical communication with the test solution 20 via a glassmembrane 15. The glass membrane 15 provides for ion exchange between afluid in the measurement chamber 12 and the test solution 20.

As with most electrochemical sensors, the glass electrode includes areference electrode 25. The reference electrode 25 is disposed within areference chamber 22 that contains a reference solution 25. Thereference solution 25 is configured to electrically communicate with thetest solution 20 through a porous fit 27, which allows for equalizationof electrical properties of the two solutions. By measuring a potentialof the measurement electrode 10 with respect to a reference potential ofthe reference electrode 22 an output potential, which is set by the ionconcentration of the test solution 20, can be communicated to the pHmeter.

The glass electrode essentially consists of four major components, theglass membrane 15, the internal electrode 10, the reference electrode 25and a glass stem. The internal electrode 10 and the reference electrode25 are both disposed in solutions. In general, the solutions are thesame solutions and may comprise solutions saturated with chloride ions,such as potassium chloride, sodium chloride etc. For best results, asymmetrical liquid cell is set up on both sides of the glass membrane.To set up the symmetrical cell, the internal fill solution in the glassand the reference fill solution are similar in their makeup. Thesymmetry is important so that the temperature curves for the twosolutions are close, thereby canceling each other's temperature effect.

Reference electrodes work like a battery with the chemical componentsproducing a predictable voltage that is also in electrical contact withthe solution being measured. The reference output is a constant voltagethus, giving the glass a reference point to distinguish changes inhydrogen ion concentration seen as a potential across the glassmembrane.

FIG. 1B is a schematic-type illustration of an electrochemical sensor.

In FIG. 1B, an electrochemical sensor 30 comprises a sensing system 35and a reference system 33. The sensing system 35 may comprise a workingelectrode and be configured to contact a test solution 45. The sensingsystem 35 comprises a chemistry configured to undergooxidation/reduction when an electrical signal is applied to the sensingsystem 35. The chemistry is selected such that the amplitude of theoxidation/reduction current and/or the potential associated with a peakof the oxidation/reduction current changes depending upon aconcentration of a particular ion/chemical species that theelectrochemical sensor is designed to detect/measure.

In the electrochemical sensor 30, the reaction from which concentrationof an ion/chemistry of interest can be determined occurs at the surfaceof the working electrode 35 of the sensing system 35. In operation, thepotential drop across the interface between the surface of the workingelectrode 35 and the test solution 45 (i.e., the interfacial potential)is controlled in the electrochemical sensor 30. However, it isimpossible to control or measure this interfacial potential withoutplacing another electrode, a counter electrode (not shown) in the testsolution 45. By using the counter electrode and the working electrode35, two interfacial potentials may be produced, neither of which can bemeasured independently. To be able to measure the interfacial potentialof the working electrode 35, a requirement for the counter electrode isthat its interfacial potential remains constant, so that any changes inthe cell potential produce identical changes in the working electrodeinterfacial potential.

An electrode whose potential does not vary with current is referred toan ideal non-polarizable electrode, and is characterized by a verticalregion on a current versus potential plot. However, there are noelectrodes that behave in this ideal manner. As a result non-idealbehavior, the interfacial potential of the counter electrode in thetwo-electrode system discussed above varies as current is passed throughthe cell. This problem is overcome by using a three-electrode system, inwhich the function of the counter electrode is divided between areference electrode 36 and one or more auxiliary electrodes (not shown).In this set-up, the potential between the working electrode 35 and thereference electrode 36 and the one or more auxiliary electrodes iscontrolled and current passes between the working electrode 35 and theone or more auxiliary electrodes. The current passing through thereference electrode 36 is reduced, as it is not desirable to havecurrent flow through the reference electrode, by using ahigh-input-impedance operational amplifier as an input to referenceelectrode 36.

In the electrochemical sensor, the reference system 33 may comprise areference chamber 34. The reference chamber 34 contains a referencesolution 39 and the reference electrode 36. The reference electrode 36is at least partially disposed within the reference solution 39. One ofthe most common reference systems is the silver-silver chloridereference system in which the reference electrode is formed from silverwith a coating of silver chloride. The reference solution for thesilver-silver chloride reference solution comprise a solution containingchloride ions, such as potassium chloride solution or sodium chloridesolution.

The redox process equation for the silver-silver chloride referencesystem is:AgCl+e-<=>Ag+Cl—

The reference solution 39 may comprise of the order of 3 Molar sodiumchloride or potassium chloride.

The potential E for any electrode is determined by the Nernst equation,which relates E to the standard potential E0 and the activities of theredox components (the standard potential is the potential of theelectrode at unit activity under standard conditions). The Nernstequation for the silver/silver chloride electrode is:

$E = {E^{0} + {\frac{RT}{nF}\ln\frac{1}{a_{{Cl}^{-}}}}}$

It is generally more convenient to consider concentrations rather thanactivities. These parameters are related by the activity coefficient g:a _(Cl) ⁻ =γ_(Cl) ⁻ [Cl⁻]

The Nernst equation can therefore be rewritten as follows:

$E = {E^{0^{\prime}} + {\frac{RT}{nF}\ln\frac{1}{\left\lbrack {Cl}^{-} \right\rbrack}}}$

Where E0′ is the formal potential and is related to the standardpotential by the equation:

$E^{0^{\prime}} = {E^{0} + {\frac{RT}{nF}\ln\frac{1}{\gamma_{{Cl}^{-}}}}}$

The standard redox potential (E0) for the silver/silver chloride redoxreaction at 25 degrees Celsius is +0.222 V (vs. NHE), whereas the redoxpotential (E) for the BASi silver/silver chloride reference electrode atthis temperature is +0.206 V (vs. NHE).

In the reference system 33, the reference chamber 39 includes a porousfit 37. The porous fit 37 ionicly conducting electrical pathway betweenthe inside of the reference chamber 36 and the test solution 45. This isnecessary in order to equalize the electrical conditions of the workingelectrode 35 and the reference electrode 36

However, Nernst equations for the silver-silver chloride referencesystem, show that variations in the chloride ion concentration in thereference chamber 34 alter the redox potential of the reference system33. In operation of an electrochemical sensor, chloride ions may passout of the reference chamber 34 through the porous fit 37 and/or thetest solution 45 may pass through the porous fit 37 into the referencechamber 34. In both of these events, the chloride ion concentration inthe reference chamber 34 is changed, and as a result the referencepotential of the reference system 33 is changed. This change inreference potential is often referred to as drift. The result ofreference electrode drift is inaccuracy in the measurement of thechemistry/ion of interest. To address reference electrode drift, theelectrochemical sensor must be regularly recalibrated. Recalibration ofthe electrochemical sensor involves an operator of the electrochemicalsensor measuring an output from the electrochemical sensor in at leastthree different solutions containing a known concentration of theion/chemistry of interest so that the output from the electrochemicalsensor can be compared to the known response that the sensor should haveproduced for the known concentrations and the sensor recalibrated toaccount for any difference found in the comparison.

By way of example, in the water industry, to meet regulations and managethe water resource multiple electrochemical pH sensors, commonly glasselectrodes, are distributed through the water management infrastructureto monitor pH. To operate the pH sensors, engineers must periodicallyvisit each of the electrochemical pH sensors and recalibrate thereference system. This is a time consuming and expensive necessity ofoperating existing electrochemical sensors. In the water industry,sensor manufacturers make sensors where recalibration may be performedevery three months or so.

However, to obtain this extended recalibration duration, the senoraccuracy is greatly diminished with resulting accuracy of the order ofplus/minus twenty percent. The long duration is made possible by usingsoftware processing where an expected/interpolated drift may beprogrammed and the reference potential adjusted accordingly. However,monitoring actual operation of the reference system is not possible, andthe accuracy of the measurements made between recalibrations, especiallywhen the time between recalibrations is long, is questionable, leadingmany sensor operators to use more frequent recalibrations. Furthermore,electrochemical sensors used in more challenging industries than thewater industry, such as in chemical processing and or chemical wastemonitoring, operate in conditions where more reactive chemicals/ions maypass through the porous frit 37 into the reference chamber 34 affectingthe reference potential generated by the reference system 33. Moreover,existing electrochemical sensors are in general “dumb” sensors thatcannot provide quality assurance, quality control data regarding sensoroperation. This need for frequent manual recalibration and/or dumboperation, mean that many electrochemical sensors, including the glasselectrode, are not capable of networked operation.

FIG. 2A illustrates an electrochemical sensor comprising an onlinecalibration system, in accordance with some embodiments of the presentdisclosure.

As illustrated in FIG., 2A, an electrochemical sensor 130 comprises asensing system 135 and a reference system 133. The sensor system 135 maycomprise a sensing/working electrode and/or the like that is configuredto provide an electronic response to an applied electronic signal thatis dependent upon a concentration of an ion/chemical species in a testsolution 145. The electrochemical sensor 130 may be an ion selectiveelectrode, such as for example a glass electrode, an amperometricsensor, a redox sensor, an ISFET and/or the like.

A processor 140, comprising processing circuitry, is configured tocommunicate with the sensing system 135 to apply a signal to the sensingsystem 135 and to process the response of the sensing system 136 to theapplied signal. To process the response of the sensing system 135, asexplained with respect to FIGS. 1A and 1B, it is necessary to provide areference potential to the processor 140 of the electrochemical sensor.

As depicted in FIG. 2A, the reference system 133 comprises a referencechamber 134. The reference chamber 134 contains a reference solution 139and a reference electrode 136. The reference electrode 136 and thereference solution 139 are configured such that the reference electrode136 generates a constant reference potential when a reference signal isapplied to the reference electrode 136. For example, the referenceelectrode 136 may comprise a silver-silver chloride electrode and thereference solution 139 may comprise a solution containing chloride ions,such as sodium chloride solution, a potassium chloride solution or thelike. In some embodiment, the reference solution 139 may comprise apaste, gel and/or the like.

In some embodiments of the present disclosure, a calibration systemcomprising a three-electrode system is provided in the reference chamber134. As depicted, the three-electrode system comprising a workingelectrode 120, counter electrode 123 and a reference electrode 126. Insome embodiments, the calibration system may comprise a single workingelectrode, a two-electrode system or a system comprising four or moreelectrodes. In some embodiments of the present disclosure, the one ofthe electrodes of the calibration system may comprise the referenceelectrode 136.

In embodiments of the present disclosure, the working electrode 120comprises a redox species 121. The redox species 121 is configured to besensitive to pH, i.e., the redox species is configured to generate aresponse signal to an applied electrical signal that varies dependingupon a pH of a solution contacted by the working electrode 120. Inembodiments of the present disclosure, the redox species 121 isconfigured to set the pH of a local environment of the referencesolution 139 in which the working electrode 120 is disposed and to sensethis set pH when a calibration signal is applied to the calibrationsystem.

Suitable redox species that are sensitive to pH/hydrogen ionconcentration and that will set the pH of the reference solution localto the calibration electrode may include, for example and withoutlimitation: anthraquinone (AQ), phenanthrenequinone (PAQ),N,N′-diphenyl-p-phenylenediamine (DPPD), anthracene, naphthaquinone,para-benzoquinone, diazo-containing compounds, porphyrins,nicotinamides, including NADH, NAD+ and N-methylnicotinamide, quinonethiol, monoquaternized N-alkyl-4,4′-bipyridinium, RuO, and Ni(OH)2,ferrocene carboxylate, and derivatives of those compounds; CO-sensitiveASMs: ferrocenyl ferraazetine disulfide; iron porphyrins; alkaline metalcation sensitive ASMs:1,1′-(1,4,10,13-tetraoxa-7,1-diazacyclooctadecane-7,16-diyl dimethyl),ferrocenyl thiol, other ferrocene derivatives containing covalentlyattached cryptands, and certain metal complexes with Fe2+/Fe3+,Co2+/Co3+, Cu+/Cu2+, ferrocenyl ferraazetine and ferrocenyl cryptands,substituted anthraquinones, mono-, di-, or poly-hydroxyl substituted AQ;mono-, di-, or poly-amino substituted AQ, ethyleneglycol orpolyethyleneglycol-modified AQ, and/or the like. The person of skill inthe art will appreciate that any redox species sensitive to pH, unlessit has been specially configured will set the local pH of the referencesolution when the redox species is oxidized/reduced because of the lowbuffering capacity of the reference solution.

As previously described, a problem with electrochemical pH sensors usinga working electrode comprising a redox active species sensitive to pH isthat when contacted with a low buffering capacity/bufferless solution,the redox active species sets the pH of the local environment of the lowbuffering capacity/bufferless solution proximal to the workingelectrode. In essence, because the solution has a low buffering capacityand/or contains no buffer, it cannot buffer the local effect of theredox active species on the solution. This results in a region close tothe redox active species having a proton pH that is dependent upon theproperties of the redox species. This problem has been identified as alimitation of the use of electrochemical sensors using redox activespecies to measure pH in low buffering capacity, bufferless solutions,such as water, seawater, sodium chloride solutions, potassium chloridesolutions and/or the like. To overcome this problem, specialized redoxchemistries have been developed that provide for accelerated response sothat the redox chemistry is sensitive to the pH of the contactedsolution not the pH of the local environment set by the redox species.

In embodiments of the present disclosure, the redox species 121 maycomprise any redox species sensitive to pH that is not configured toovercome the effect of localized pH setting in low bufferingcapacity/bufferless solution. In preferred embodiments, the redoxspecies 121 is selected to set a pH of the local environment of thereference solution 139 to a pH value that is greater than one pH unitabove or below pH 7. Applicant has found that calibration processing isoptimized when the redox active species 121 sets a pH that is not at orclose to pH 7. In some embodiments, the redox active species 121 maycomprise an acid, a base, acid moieties or base moieties.

Applicant has found that nearly all of the generally used redox speciessensitive to pH can be used to set the pH of the local environment andto produce a response to this set pH, as very few of the redox activespecies sensitive to pH provide for overcoming the low buffer capacityissue. By way of example, Applicant has found that anthraquinone and itsderivatives, some of the most commonly used redox species sensitive topH, can be used as the redox active species 121. By way of example,anthraquinone sets the pH of the local environment of the referencesolution to a pH value of about 10.

For embodiments of the present disclosure, the redox species 121 isconfigured to be sensitive to pH and to set a local pH of the referencesolution 139 proximal to the working electrode 120. These features ofthe redox species 121 and the fact that the redox species 121 is notsensitive to chloride ion concentration, provide that a response of theredox species will be a constant value whatever the changes to thechloride concentration.

In embodiments of the present disclosure, the reference solution 139comprises a low buffering capacity/bufferless solution. As noted above,the most common reference solutions for the reference system 133comprise sodium chloride, potassium chloride, or the like. Bothpotassium chloride and sodium chloride solutions comprise low bufferingcapacity/bufferless solutions. Potassium chloride (KCl) referencesolutions do not comprise any buffer in the reference solution, so arewell suited to use in embodiments of the present disclsoure.

In some embodiments of the present disclosure, a calibration processor150 may apply an electrochemical/voltammetric signal to the workingelectrode 120. The voltammetric signal may comprise a varying electronicsignal, such as a square wave voltammetric signal, a ramped voltammetricsignal and/or the like. In embodiments of the present disclosure, inwhich the calibration system comprises a three-electrode system, thevoltammetric signal may be swept across between the working electrode120, the counter electrode 123 and the reference electrode 126.

In response to the application of the voltammetric signal, the workingelectrode generates an electrochemical/voltammetric response. Thisresponse is dependent upon the oxidation/reduction current produce bythe redox species 121. Since the redox species 121 is sensitive to pH,the oxidation/reduction current produced by the redox species 121 willdepend upon the pH of the reference solution 139. In embodiments of thepresent disclosure, because the redox species 121 is configured to setthe pH of the reference solution proximal to the working electrode120/redox species 121, the voltammetric response will have constantfeatures, e.g., a peak potential corresponding to a peak/singularity inthe oxidation/reduction current, corresponding to the set pH. Inembodiments of the present disclosure, the voltammetric response may beprocessed by the calibration processor into a voltammogram and/or into arepresentation showing working electrode potential versus redox current.

In embodiments of the present disclosure, the redox species 121 and thereference solution 139 are selected so that the redox species 121, whichis sensitive to pH, sets the pH of the reference solution 139 local tothe redox species 121. In this way, in embodiments of the presentdisclosure, the peak potential in the voltammetric response is aconstant. In some embodiments of the present disclosure, the peakpotential is used by the calibration processor 150 and/or the processor140 to calibrate the reference potential of the reference system 133.Merely, by way of example, in some embodiments, a difference between thereference potential and the peak potential may be determined and storedby at least one of the calibration processor 150 and/or the processor140. This difference may be: measured when the electrochemical sensor130 is initially deployed by a user using measurements from thereference system 133 and/or the calibration system; may be calculatedfrom empirical calculations; may be determined from batch measurementsmade on one or more electrochemical sensors in a manufactured batch ofelectrochemical sensors; and/or the like.

Merely by way of example, in some embodiments, the peak potential forthe redox species 121 for the set pH generated in the local environmentof the reference solution 139 may be measured, calculated, determinedfrom experiments/testing and/or the like and may be entered into thecalibration processor 150 and/or the processor 140. Subsequently, when auser calibrates the reference system 133, the calibration processor 150and/or the processor 140 may record the difference between the peakpotential and the reference potential.

In operation, as described above, the reference potential of theelectrochemical sensor 130 drifts as a result of changes to thereference solution 139, such as reduced concentration of chloride ionsin the reference solution 139. In embodiments of the present disclosure,a voltammetric signal is repeatedly applied to the calibration system togenerate a peak potential. This peak potential and the known/recordeddifference between the peak potential and the reference potential, asdescribed above, is used to process a calibration factor to calibrateany changes in the reference potential due to changes in the referencesolution 139. The periodicity of the applied voltammetric signals maydepend upon the use of the electrochemical sensor 130 and may be set bythe calibration processor 150 and/or the processor 140, set by the user,set by the manufacturer and/or the like. For example, where theelectrochemical sensor 130 comprises a glass electrode and is used fordeployments of weeks/months, the periodicity may be of the order ofhours or days. In sensor deployments of hours or days, the periodicitymay be of the order of seconds, minutes or hours.

In embodiments of the present disclosure, by repeatedly measuring thepeak potential associated with the redox species 121, the calibrationsystem is configured to provide for online calibration of the referencepotential of the reference system 133. As noted previously, thereference system 133 comprises a porous frit 137 that provides anionicly conducting electrical pathway between the inside of thereference chamber 134 and the test solution 145. This porous frit 137allows for dilution of the reference solution 139 and/or entry ofchemical species into the reference solution 139. In tests, applicanthas found that the calibration system produces a stable peak potentialthat is constant and dependent only upon the set pH in referencesolutions less than 1 molar sodium or potassium chloride up to about 10molar sodium or potassium chloride. This illustrates that embodiments ofthe present disclosure can be used in most commercially availablereference systems.

Applicant has also found that the peak potential of the calibrationsystem is stable/constant when the reference solution 139 has beendiluted by up to 50%. Surprisingly, applicant has found that the peakpotent of the calibration system is stable/constant when: acids, such ashydrochloric acid; bases, such as sodium hydroxide; carbonates, such ashard water; reactive chemistries, and other reactive chemistries areadded to the reference solution 139. In this disclosure, reference ismade to a peak potential produced by the working electrode 120 inresponse to an applied voltammetric signal. This peak potentialcorresponds to a peak/trough in the redox current produced by the redoxspecies 121. In some embodiments, other features in the voltammetricresponse may be used instead of the peak potential for calibrating thereference potential. For example, rather than the peak potential thepotential corresponding to another feature in the voltammetric responsemay be used, such as potential/position corresponding to the greatestrate of change in the response and/or the like. In embodiments of thepresent disclosure peak picking algorithms or the like may be used toidentify a peak in the voltammetric response. In some embodiments,because the peak potential for the redox species 121 when contacted withthe reference solution 139 is known/can be calculated, thisknown/calculated peak potential may be used to process the voltammetricresponse when determining the peak potential from the voltammetricresponse.

FIG. 2B illustrates an electrochemical sensor comprising a calibrationsystem in accordance with some embodiments of the present disclosure.

The electrochemical sensor comprises a sensor electrode 150 and areference system 160. The reference system 160 comprises a referencechamber 162 containing a reference solution 165 and a referenceelectrode 151. The reference electrode 151 is at least partially incontact with the reference solution 165. The reference solution 165 maycomprise sodium chloride, potassium chloride etc.

The sensor electrode 150 may comprise a glass electrode, with a glassmembrane etc. The sensor electrode 150 is configured to be in electricalcommunication/contact with a test solution 155. The reference solution165 is configured to be in electrical communication with the testsolution 155 via a porous fit 167. The reference electrode 151 isconfigured to generate a reference potential for the electrochemicalsensor.

A typical reference electrode is the silver chloride electrode. Thesilver chloride reference electrode functions as a redox electrode wherean equilibrium is provided between the silver metal of the referenceelectrode and its salt, silver chloride, of the reference solution. Thereference potential is constant given the constant condition of thereference electrode and the reference solution. However, the referencepotential will drift if the concentration of the silver chloride in thereference chamber changes. Since the reference solution is in fluidcommunication with the test solution through the porous frit 167 theconcentration will change during use of the electrochemical sensor.

In embodiments of the present disclosure, a calibration electrode 163 ofan additional electrochemical cell is placed inside the existingreference electrode chamber. The electrochemical cell may comprise acounter electrode 161 and an auxiliary electrode 164. In someembodiments, the reference electrode 151 may also be used as thereference electrode/auxiliary electrode 164 in the electrochemical cell.The calibration electrode 163 is the working electrode of theelectrochemical cell and comprises a redox species that is immobilizedon the calibration electrode 163 and both redox active, sensitive to pH,and also controls the local environment of the reference solution 165close to the surface of the calibration electrode 163.

In some embodiments, the immobilised redox species may be solvent castonto, electro-polymerised on, or immersed within the calibrationelectrode 163. The redox couple bound within the polymeric layer acts asa new stable redox couple, whilst the layers ability to control thelocal environment acts as a second layer to protect from any variationsobserved in the bulk solution in which the electrode is immersed. Thisadditional electrochemical circuit acts as a means of correcting anydrift observed in the reference electrode in-situ.

In some embodiments, the device runs under potentiometric sensing modebetween the analyte sensing electrode and the reference electrode. Thecell periodically runs a voltammetric sweep against the workingelectrode housed behind the porous frit. The potential of the redoxactive species immobilized on the working electrode is used to correctany drift occurring in the reference electrode. In some embodiments, thefrequency of voltammetric sweep depends on the application for which theelectrochemical sensor is deployed.

FIG. 2C depicts a schematic representation of a sensor output from anelectrochemical sensor comprising a calibration system, in accordancewith some embodiments of the present disclosure, using variousvoltammetric sweep profiles. In the depicted configuration, thecalibration system can provide QA/QC of the sensor's referenceelectrode. The depicted sensor includes an integrated electrochemicalcell and potentiostat, and the system uses a voltammetric signal fromthe potentiostat to produce a sweep between the reference electrode andthe calibration electrode in the integrated electrochemical cell togenerate a potential that may be used for QA/QC of the referenceelectrode during operation of the sensor. The sensor may in someembodiments run using a modified bi-potentiostate system. Such a set-upobviates the need for reference sensor calibration prior to deploymentas is the case with current commercial ISE's. In embodiments of thepresent disclosure, prior to deployment, the internal circuit can bemeasured and this may be used to set the parameters for the referencesystem.

Embodiments of the present invention may use the following chemicalstructures on the working electrode, in which the redox active componentof molecule has carboxylic, sulfonic and/or amino moieties.

In certain cases such as that of salicylic acid (top left structure) aredox active pH active polymeric layer can be formed containingcarboxylic acid moieties, which in the proposed set-up the redox activecomponents only observes a pH of solution consistent with that of thepKa of the molecule.

In certain embodiments, the working electrode in the new electrochemicalcell can have a single layer in which the redox active component hasmoieties attached to control the local environment directly and in otherembodiments the working electrode system can have a dual layer in whichthe redox active components are separated/independent from the speciesused to control the local environment. In other embodiments, the redoxactive species used within the working electrode system by virtue of itsoxidation or reduction can control the local environment to the surfacethrough the release/loss or gain of protons, cations or anions.

FIG. 3 is a flow-type illustration of a method for online calibration ofreference electrode of an electrochemical sensor, in accordance withsome embodiments of the present disclosure.

In 310, a calibration electrode is provided to contact a referencesolution of a reference system of an electrochemical sensor. Thereference system comprises a reference chamber containing the referencesolution and a reference electrode. The reference electrode may comprisea metal, carbon or the like and the reference solution may comprise asalt, such as sodium chloride, potassium chloride or the like.

The reference electrode comprises a redox active species that issensitive to pH/proton concentration. The reference solution comprises abufferless/low buffer capacity solution, such as potassium chloride,sodium chloride and/or the like. The redox active species is configuredto set a pH of the reference solution local to/proximal to the redoxactive species. The redox active species may be immobilized on thecalibration electrode, covalently bound to the calibration electrodeand/or the like. The calibration electrode may comprise carbon, a carbonderivative, a metal, and/or the like. The redox active species isselected not to be sensitive to chloride ions/chloride ionconcentration.

In 320, a voltammetric signal/a potential sweep is applied to thecalibration electrode. The calibration electrode may be part of acalibration system comprising a counter electrode and the voltammetricsignal may be swept between the calibration electrode, which maycomprise a working electrode and the counter electrode. The voltammetricsignal may comprise a potential sweep, such as a square wave, a rampedsignal/wave and/or the like.

The applied voltammetric signal causes the redox active species toundergo oxidation/reduction. Since the redox active species is sensitiveto pH/proton concentration of the reference solution it is in contactwith, the oxidation/reduction current will be determined by the pH ofthe reference solution. Moreover, because the reference solution is abufferless/low buffer solution, the pH of the reference solutionproximal to the redox species is set by the properties of the redoxactive species. In some embodiments, the redox active species maycomprise an acid, a base, acid moieties, basic moieties and/or the like.In other embodiments, the redox active species may trigger the pH of thelocal environment by donating or consuming protons when anelectrochemical/voltammetric signal/potential sweep is applied to theredox active species. For example, in some embodiments, the redox activespecies may comprise anthraquinone and the anthraquinone may set thelocal pH of the reference solution to a pH of approximately 10 byconsuming protons. Because the redox active species is selected to setthe pH of the reference solution local to the redox active species, thepeak potential of the reduction/oxidation current will be determined bythe pH set by the redox active species.

In 330, a voltammetric response of the calibration electrode to theapplied voltammetric signal is processed to determine a calibrationpotential. In some embodiments, the calibration potential is the peakpotential produced by the redox active species in response to theapplied voltammetric signal. In some embodiments, the voltammetricresponse may be a current versus potential and the voltammetric responsemay contain a peak/singularity corresponding to a peak/trough in theredox current of the redox active species. The potential correspondingto this peak/singularity in the redox current is the peak potential. Insome embodiments, other features of the peak/singularity may be used asa calibration potential. Because the redox current and the peakpotential for the redox active species correspond to the pH of referencesolution seen by the redox active species, and because the redox activespecies sets the pH of the reference solution local to the redox activespecies, the redox current and the peak potential are a constant. Inembodiments of the present disclosure, the redox active species isselected such that its redox response is not affected by chlorideconcentration in the reference solution. This and the fact thatapplicant has found that the response of the redox active species in thepresent arrangement is not sensitive to acids, basis, hard water,reactive chemistries and/or the like in the reference solution meansthat the calibration electrode provides a constant/stable response tothe applied voltammetric signal over a wide range of challengingchemical applications.

In 340, the calibration potential is used to calibrate the referencepotential of the electrochemical sensor. In some embodiments, adetermined difference between the calibration potential and thereference potential is used for online calibration of theelectrochemical sensor. For example, the reference potential of theelectrochemical sensor may be determined when the sensor is new, beingdeployed, being calibrated and/or the like. At this time, a differencebetween the reference potential and the calibration potential may bedetermined and stored. The calibration potential itself may becalculated, measured, determined from experimentation, be measuredduring manufacture of the electrochemical sensor and/or the like. Inoperation when the electrochemical sensor is being used, a periodicmeasurement of the calibration potential is processed and compared tothe reference potential. If the difference between the calibrationpotential and the reference potential has changed with respect to thestored difference, the reference potential is recalibrated to accountfor this change. In this manner, the reference potential for theelectrochemical sensor can be recalibrated online, while theelectrochemical sensor is deployed and making measurements.

Polymer Layer

Rigorous testing of the redox active component in the presence of anumber of different anions and cations showed an independence of thepeak potential with ion concentration and charge. However, due to thenature of the reference system—controlling the local environment throughperturbing the pH of the solution—in media of high buffer concentration,the measurement proves problematic as the local environment is swampedby the buffer and hence the local pH will be that of the bulk solution.Therefore rather than acting as a reference compound, the redox activespecies will have a redox potential dependent on the pH of the bulksolution. FIG. 4 details an AQ electrode in three IUPAC buffered media;pH 4 phthalate, pH 7 phosphate and pH 10 carbonate buffer. It is evidentthat under these conditions the AQ modified electrode no longer acts asa reference system. It is thus desired to minimize this pH shiftbehaviour seen in highly buffered media.

The use of polymeric layers in electrochemical sensors to increaselifetimes or to increase selectivity for specific analytes througheither molecular (host/guest) or charge (anion/cation) interactions iswell known. Examples of these systems are vanillomycin used topreferentially determine potassium over calcium, or anionic films(Nafion) to selectively retard a number of biological redox activeinterferences (ascorbic acid, uric acid etc . . . )

In the proposed invention, the use of polymeric layers to impart furthercontrol on the local environment is proposed. The polymeric layerprovides a constrained environment in which the electrochemistry canoccur and within a hindered diffusion regime to the bulk solution. Thiswill minimise the effect of the bulk solution on the local environmentperturbation, and thus allows greater control over the localenvironment.

FIG. 5 details two types of electrode set-ups. FIG. 5A incorporates fourcomponents; the electrode, the electroactive system, the polymeric layerand the bulk solution. In this set-up the electroactive species iseither chemically or physically immobilized onto the surface of theelectrode, and the polymer is then immobilised upon the surface. In suchinstance the polymer can be immobilised onto the electrode surfacethrough solvent casting, or electrochemical or chemically polymerizedonto the surface in-situ. The fourth component is the bulk solution.FIG. 5B details a three component system involving a bulk modifiedelectrode, a polymeric layer and the bulk solution. In this case theconducting electrode contains the redox moiety within the bulk of theelectrode. The polymer is immobilised on the electrode surface throughsolvent casting or electrochemical or chemically polymerization onto thesurface in-situ. It should be noted that in certain embodiments, one ormore polymeric layers can be placed onto the electroactive surface, toprovide further control of the surface.

In certain cases the polymer can be anionically charged (Nafion, anionicpolyacrylamides, salicylic acids), cationically charged (e.g.,polylysine, polyornithine, polybrene, polyethyleneimine, cyclodextrin,chitosan, histone, collagen) or neutral (polyacrylamides, polyacrylates,polysaccharides, polysulfone, polyaniline, polythiophene etc . . . )depending on the pH of the bulk solution.

It should be noted that the charge of the polymer may be dependent uponthe bulk solution which the polymer is in. For example, Polyaniline maybe naturally charged in a low pH solution, whilst being neutral athigher pH solution. A further example is that Nafion may be neutral whenin a solution below a pH of approximately 2 whilst being anionicallycharged when in a solution above a pH of approximately 2.

FIG. 6 details the electrochemical system shown to be pH responsive inbuffered media (FIG. 4 ) with a Nafion coating, when placed in 3M KCl,pH 4, pH 7, pH 10 buffers and a hard water sample with 1M KCl present.It can be clearly seen that unlike the data presented in FIG. 4 , thepeak potential is now independent of pH of the sample, showing that thenafion layer imparts further control on the environment local to theelectrode surface. The pH of the surface in this case is set by theuptake of protons local to the electrode surface due to the reduction ofthe anthraquinone moiety.

FIG. 7 details the response of a) polyaniline, b) cellulose acetate andc) an electropolymerised salicylic acid coated electrode when placed in3M KCl and pH 10 solution. Minimal shift is observed between thebuffered and unbuffered responses, showing the efficacy of using theselayers to provide stability on the peak potential in the various media.In these systems, both polyaniline and cellulose acetate will have aneutral charge, whilst the electropolymerised salicylic acid will havean anionic charge.

Multiple Cycles and Time Between Scans

To understand how the polymeric layer imparts stability on the electrodelayer, various experiments were conducted to understand how theimposition of electrode potential effects the equilibrium of the system.To achieve this a Nafion coated electrode was placed in a pH 10 bufferedsolution and the potential was cycled 20 times continuously with nodelay between scans. The electrode was then left under quiescentconditions for a known period prior to the potential being cycled again.This process was then repeated. FIG. 8 details the plot of peakpotential against time showing the peak potential measured for thefirst, 10th, 15th and 20th scan. It can be seen that repetitivelyscanning the potential shifts the peak potential to a lower value.Furthermore, the data shows when a short time period is left between theconsecutive scans (ca. 2-50 mins, FIG. 8A) the peak potential observedon the first scan does not return to the peak potential observed on scan1 at 0 minutes. However, when a longer time period is left between thescans (80 mins, FIG. 8B), the peak potential measured on the first scanreturns to the starting potential, indicating the system has returned toequilibrium. The data can be explained as, following the first 20initiation scans, the potential is lowered due to the electrochemicalprocess consuming protons at the electrode surface, after each scan thelocal pH becomes more alkaline as further protons are consumed. Thepolymeric layer hinders the rate of proton transfer from the bulksolution to the electrode surface, and as such the equilibrium betweenthe bulk and electrode surface cannot be maintained. The localenvironment pH is then controlled by the consumption of protons at theelectrode surface. Under quiescent conditions, for short periods oftime, the local pH is still not consistent with the bulk pH, asdiffusion is slow to the electrode surface through the polymeric layerand hence the peak potential is lower than at scan 1 time 0. Only aftera significant period of time between scans (ca. 80 mins) doesequilibrium between the local environment and the bulk solution return.

While the principles of the disclosure have been described above inconnection with specific apparatuses and methods, it is to be clearlyunderstood that this description is made only by way of example and notas limitation on the scope of the invention.

What is claimed is:
 1. An online calibration system for anelectrochemical sensor, comprising: a calibration electrode in areference solution, the calibration electrode comprising: a redoxspecies, wherein: the redox species is configured to set a pH of thereference solution in a local environment proximal to the calibrationelectrode; the redox species is configured to undergo oxidation and/orreduction when an electrochemical signal is applied to the calibrationelectrode; and the redox species is sensitive to pH and generates aresponse to the applied electrochemical signal that is dependent on thepH of the local environment proximal to the calibration electrode, and apolymeric layer positioned between the redox species and the referencesolution.
 2. The online calibration system according to claim 1, whereinthe polymeric layer is anionically charged.
 3. The online calibrationsystem according to claim 1, wherein the polymeric layer is formed ofNafion and/or anionic polyacrylamides and/or salicylic acid.
 4. Theonline calibration system according to claim 1, wherein the polymericlayer is cationically charged.
 5. The online calibration systemaccording to claim 1, wherein the polymeric layer is formed ofpolylysine and/or poly ornithine and/or polybrene and/orpolyethyleneimine and/or cyclodextrin and/or chitosan and/or histoneand/or collagen.
 6. The online calibration system according to claim 1,wherein the polymeric layer is neutral.
 7. The online calibration systemaccording to claim 1, wherein the polymeric layer is polyacrylamidesand/or polyacrylates and/or polysaccharides and/or polysulfone and/orpolyaniline and/or polythiophene.
 8. The online calibration systemaccording to claim 1, wherein the redox species is chemically orphysically immobilized on a surface of the calibration electrode.
 9. Theonline calibration system according to claim 8, wherein a polymer of thepolymeric layer is immobilised over the redox species.
 10. The onlinecalibration system according to claim 1, wherein the calibrationelectrode contains the redox species in a bulk of the calibrationelectrode.
 11. The online calibration system according to claim 10,wherein the calibration electrode contains the redox species throughoutan entire structure of the calibration electrode.
 12. The onlinecalibration system according to claim 1, wherein a polymer of thepolymeric layer is immobilised onto the surface of the calibrationelectrode.
 13. The online calibration system according to claim 12,wherein the polymer is immobilised through solvent casting orelectrochemical polymerization or chemical polymerization.
 14. Theonline calibration system according to claim 1, wherein the redoxspecies is configured to consume or donate protons when theelectrochemical signal is applied to the calibration electrode.
 15. Theonline calibration system according to claim 1, wherein theelectrochemical signal comprises a potential sweep and/or a voltammetricsignal.
 16. The online calibration system according to claim 14, whereinthe redox species comprises anthraquinone or a derivative thereof. 17.The online calibration system according to claim 1, wherein thereference solution comprises sodium chloride or potassium chloride. 18.The online calibration system according to claim 1, further comprising:a processor in communication with the calibration electrode and areference electrode of the electrochemical sensor and configured to usethe electrochemical response to calibrate a reference potential of thereference electrode.
 19. The online calibration system according toclaim 18, wherein the electrochemical response comprises a peakpotential corresponding to a maximum of an oxidation current or aminimum of a reduction current produced by the redox species.
 20. Theonline calibration system according to claim 1, further comprising: apotentiostat configured to generate the electrochemical signal.
 21. Theonline calibration system according to claim 1, wherein theelectrochemical signal comprises one of a square wave, a ramped waveand/or a pulsed wave.
 22. The online calibration system according toclaim 1, further comprising: either separate counter and referenceelectrodes and/or a combined counter-reference electrode.
 23. The onlinecalibration system according to claim 22, wherein the electrochemicalsignal is applied between the calibration electrode and either theseparate counter and reference electrodes and/or the combinedcounter-reference electrode.
 24. The online calibration system accordingto claim 1, wherein the calibration electrode comprises at least one ofa microelectrode and a microelectrode array.
 25. The online calibrationsystem according to claim 1, wherein the calibration electrode and thereference solution are disposed within a reference cell of theelectrochemical sensor.
 26. The online calibration system according toclaim 25, wherein the reference cell comprises a frit configured tocontact a fluid being sensed by the electrochemical sensor.
 27. Anelectrochemical sensor comprising the calibration system according toclaim
 1. 28. The electrochemical sensor of claim 27, wherein theelectrochemical sensor comprises one of a glass electrode, an ISFET or apotentiometric sensor for measuring concentration or presence of aspecific ion.
 29. A method for online calibration of an electrochemicalsensor, the method comprising: placing a calibration electrode in areference solution of the electrochemical sensor, wherein thecalibration electrode comprises a redox species configured to control pHof a local environment proximal to the electrode and a polymeric layerpositioned between the redox species and the reference solution;applying a voltammetric signal to the calibration electrode to produceoxidation and/or reduction of the redox species; and using a calibrationpotential corresponding to a feature of an oxidation or a reductioncurrent generated by the redox species to calibrate a referencepotential of the electrochemical sensor.
 30. The method according toclaim 29, wherein using the calibration potential to calibrate theelectrochemical sensor comprises using a difference between thereference potential of a reference electrode and the calibrationpotential to calibrate the reference potential.
 31. The method accordingto any one of claim 29, wherein applying a voltammetric signal to thecalibration electrode comprises applying a voltammetric sweep across thecalibration electrode and a calibration reference electrode.
 32. Themethod according to claim 31, wherein the calibration referenceelectrode comprises a reference electrode of a potentiometric sensor.33. The method according to any one of claim 32, wherein thepotentiometric sensor is calibrated at the same time as it is being usedto sense properties of fluid.
 34. The method according to claim 29,wherein the reference solution is contained in a reference cell of theelectrochemical sensor.
 35. The method according to claim 29, whereinthe voltammetric signal is applied periodically to the calibrationelectrode.
 36. The method according to claim 29, wherein the redoxspecies is configured to set the pH of the local environment to a pHvalue less than 6 or a pH value greater than
 8. 37. The method accordingto claim 29, wherein the reference solution comprises one of: potassiumchloride and sodium chloride.
 38. The method according to claim 29,wherein the voltammetric signal is applied multiple times prior to usingthe calibration potential to calibrate a reference potential of theelectrochemical sensor.
 39. The method according to claim 38, whereinthe voltammetric signal is applied at least 20 times prior to using thecalibration potential to calibrate a reference potential of theelectrochemical sensor.
 40. The method according to claim 38, whereinthe voltammetric signal is applied 5 or 10 or 15 or 20 times prior tousing the calibration potential to calibrate a reference potential ofthe electrochemical sensor.
 41. The method according to any one of claim29, wherein a wait time is established between the applying of thevoltammetric signal and the application of a next voltammetric signal.42. The method according to claim 41, wherein the wait time is 50minutes or more.
 43. The method according to claim 42, wherein the waittime is 80 minutes or more.